Polymerase Chain Reaction, or PCR, allows an initial amount of DNA in a suitable reaction mix to be copied many times, thus increasing the amount of that DNA in the sample. PCR consists of repeated cycles of a denaturing phase wherein the DNA double helix is separated, an annealing phase wherein primers bind to the DNA target, and an extension phase wherein the primers are elongated by DNA polymerase, thus selectively amplifying the target DNA.
Real time quantitative PCR (qPCR) is a widely used technique based on monitoring the PCR reaction during its progress. This can be accomplished thanks to suitable fluorescent probes, which bind either to the target DNA or to the duplex DNA, and thus allow signal increases as the reaction proceeds and the DNA is amplified.
Multiplex PCR is the simultaneous amplification of more than one target sequence in a single reaction, and multiplex PCR can also be performed in real time PCR assay format. Known multiplex real-time PCR platforms use probe-based assays, in which each target DNA has a specific probe labeled with a unique fluorescent dye, resulting in different observed colors for each assay.
It is also possible to use two labels per probe, with a fluorescent dye at one end and a quencher at the other end. This technology is called Molecular Beacons®, commercially available from SIGMA-ALDRICH®, and described in U.S. Pat. Nos. 5,925,517, 6,034,130, 6,103,476, 6,150,097, and 6,461,817.
Molecular Beacons® are hairpin shaped molecules with an internally quenched fluorophore, whose fluorescence is restored when it binds to a target nucleic acid sequence and thus unwind the hairpin, separating the two labels, which then no longer quench each other, and resulting in a signal increase.
The TaqMan® probe is analogous to the molecular beacon probe, having two labels in close proximity that quench each other when in a hairpin loop formation. However, the TaqMan® probe hybridizes to target DNA between the pair of primers, so the 5′ exonuclease activity of the DNA polymerase cleaves off the fluorophore, allowing an increase in fluorescence.
Scorpion® probes are another real time PCR probe having dual labels that quench each other when in a hairpin loop formation. Scorpions® contain a PCR primer covalently linked to a hairpin probe with dual quenched labels. During a PCR reaction, the fluorophore and quencher are separated, which leads to an increase in light output from the reaction tube. The important difference between this technology and TaqMan® is that the probe and the target in a Scorpion® reaction are in the same molecule such that signal generation is via a uni-molecular rearrangement. In contrast, a TaqMan® reaction requires a bi-molecular collision.
Ideally, a real-time multiplex PCR should be able to detect, differentiate, and provide a quantitative result for many different targets without any single target influencing the detection of one of the others (cross-talk) and without loss of sensitivity. The ideal platform would also allow both multiplexed assays and massively parallel assays, so that a great many assays can be performed at once. Further, the platform would require only small volumes and provide a reproducible result very quickly, and microfluidic platforms have been developed to fill this need.
However, known microfluidic platforms do not provide sufficient parallelism (i.e. number of reaction chambers working in parallel), combined with small volumes of reagents and sample (e.g., less than 300 nl per chamber), high versatility, ease of use and cost effectiveness, all necessary in order to improve the manufacturing scalability in large volumes of the fabricated disposable devices and also to reduce the overall cost of the envisaged solution, including the cost of both the disposable device and reagents.
Moreover, known microfluidic devices having a plurality of reaction chambers or wells formed in a same substrate, still suffer from cross contamination between the wells (fluid/reagent within one well mix with the fluid/reagent from another well). This drawback is identifiable in known microfluidic devices irrespectively of the particular use of such microfluidic devices.